A variety of research applications require the use of cultured cells. However, it often is difficult to obtain well embedded monolayers of cultured cells for ultrastructural analysis, especially if sections parallel to the cell monolayer are desirable. Previously published techniques for obtaining plastic embedded cell monolayers suitable for thin-sectioning include: (1) precoating the glass coverslip (on which the cultured cells are adherent) with some substance that will act as a fracture plane for removal of the coverslip after polymerization of the epoxy resin (Sheffield, 1965); (2) growing cells on surfaces other than glass coverslips that will be compatible with dehydration in ethanol or acetone (Egeberg, 1965; Dalan and Nevalainen, 1968; Miller and Jones, 1987); or (3) etching coverslips with a corrosive such as hydrofluoric acid to remove the coverslip from the polymerized epoxy resin (Moore, 1975). In this report, we present a method for removal of coverslips following polymerization of the epoxy resin that is simple, economical, and highly repeatable. Most importantly, the ultrastructure of the cultured cells is not compromised.
Cultures of cells (in this case P-19 cells) were grown on collagen-coated glass coverslips and routinely processed in aluminum weighing pans for electron microscopy, including primary fixation in glumaldehyde-paraformaldehyde, postfixation in osmium tetroxide, en bloc staining with uranyl acetate, and dehydration in a graded series of acetones prior to infiltration with Spurr's low viscosity resin. Dehydration may be carried out in ethanol and the cells embedded in Epon resin, with equally satisfactory results. To embed the cells, the coverslips with their adherent cell monolayer were inverted and placed over a rubber disc embedding mold (Ladd Research Industries, Inc.) filled with epoxy resin (Fig. la). Care was 0 1988 ALAN R. LISS, INC.